Method for synthesis of the second strand of cDNA

Abstract

Synthesis of the second strand of cDNA using pol ∝-primase is provided. Pol ∝-primase is used to initiate primers on a cDNA strand de novo, followed by extension of the primer with pol ∝-primase, a second DNA polymerase or a combination thereof. In a preferred embodiment, pol ∝-primase extends the primer, followed by addition of a second DNA polymerase, preferably Klenow fragment.

Description

FIELD OF THE INVENTION[0001]The present invention relates to the field of nucleic acid synthesis, more particularly the synthesis of a double-stranded cDNA from mRNA (“RNA”).BACKGROUND OF THE INVENTION[0002]Standard protocols exist in the literature for generating double-stranded cDNAs from cellular mRNA. See, e.g., Sambrook, Fritsch, and Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989.[0003]Generally, to prepare a double stranded cDNA from RNA, a first cDNA strand is synthesized using RNA as a template, resulting in an mRNA:DNA hybrid. The mRNA is then degraded by enzymes such as RNAse H. The second cDNA strand is then synthesized by using the first cDNA strand as a template.[0004]Many methods exist for the synthesis of the second strand, but these methods have disadvantages. One method involves self-priming. The 3′ termini of single-stranded cDNAs form hairpin structures that can be used to prime synthesis. In this method...

AI Technical Summary

Benefits of technology

[0012]A preferred method of extending the primer is by having pol α-primase extend the primer initially followed by addition of a second DNA polymerase with high processivity. A preferred second DNA polymerase is Klenow fragment.

Problems solved by technology

Many methods exist for the synthesis of the second strand, but these methods have disadvantages.

A self-primed synthesis of the second strand is a poorly controlled reaction, and the subsequent cleavage of the hairpin structure can lead to the loss or rearrangement of sequences corresponding to the 5′ terminus of the mRNA.

RNAase H produces nicks and gaps in the RNA strand of the hybrid, creating a series of RNA primers that can be extended by E.coli DNA polymerase I. With nick-translation, the amount of RNAase H activity must be closely monitored and controlled; there must be sufficient hydrolysis of the RNA to generate large gaps, but too much degradation will result in few or no primers for synthesis.

The ligation reaction is often inefficient, especially when a gap exists between the two DNA fragments.

A problem with random hexamer priming is that the reaction is performed at 37° C., which is above the Tm for annealing of the hexamers to the DNA template.

Hence, secondary structure can have a strong influence as to where synthesis is initiated and terminated, which can result in an incomplete synthesis of the second strand.

Furthermore, random priming requires the synthesis of the random primers on a DNA synthesizer followed by exogenous annealing, which can be burdensome.

A problem with homopolymeric tailing by terminal transferase is that the reaction can not be closely controlled.

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